Bone loss is an in oral health problem which occurs in both periodontal disease and the alveolar ridges of edentulous patients. We are therefore interested in factors which have been shown to increase bone resorption. Originally, epidermal growth factor (EGF) was found to cause accelerated tooth eruption in neonatal mice. In addition, EGF causes increased resorption in long bone and calvarial culture systems and the EGF receptor (EGFR) is located on preosteoblasts surrounding the periodontal ligament in rats. It is possible that EGF plays a role in tooth eruption and control of alveolar bone deposition and loss. The EGFR, as well as receptors for other bone resorptive agents, such as parathyroid hormone, are located on osteoblasts. In order to understand how osteoblasts respond to these factors we plan to study signal transduction mechanisms of bone resorptive agents in osteoblastic cells. In the different cells which have been studied, EGF causes an increase in phosphatidylinositol hydrolysis and a subsequent increase in intracellular free calcium. The EGFR has been reported to activate phospholipase C (PLC) isozymes by a number of mechanisms in different cell types. The EGFR has been associated with PLC activation via pertussis toxin-sensitive G proteins and it can directly phosphorylate the PLCgamma isozyme. Although the toxin-insensitive G proteins are known to activate PLCgamma, their role in EGF activation of PLC remains to be examined. We have found that primary culture osteoblasts isolated from neonatal rat calvaria, and the clonal osteoblastic osteosarcoma cell lines G292 and SaOS-2 have differences in their mitogenic response to EGF. All of these cells have receptors for this growth factor, therefore we assume that there must be differences in their signal transduction pathways. It is the aim of these studies to examine early events in the EGF signal cascade in primary culture osteoblastic cells. In specific aim one, it will determined if EGF activates either pertussis toxin-sensitive, cholera toxin-sensitive or toxin-insensitive G proteins using a radiolabeled GTPgamma-S binding assay. In specific aim two, EGF activation of PLC by toxin-sensitive and toxin-insensitive G proteins will be assessed by coimmunoprecipitation experiments. These studies will look at changes in EGFR-G protein complexes and G-protein-PLC complexes as a function of EGF stimulation. In specific aim three, it will be determined if the EGFR directly phosphorylates PLCgamma. These experiments will look for EGF stimulated EGFR PLCgamma complexes as well as an increase in PLCgamma tyrosine phosphorylation. These studies will help to understand how resorptive agents act via osteoblasts to bring about bone loss.